The long-term objective of this application is to develop a detailed understanding of the molecular and structural basis for red cell membrane mechanical function in health and disease. The principal aim of this application is to critically define the origins of red cell membrane material behavior at the molecular level by combining new functional and structural information on individual proteins with detailed biophysical characterization of red cells. To achieve the stated objective, we propose the following four specific aims: 1) Explore the hypothesis that dissociation of integral proteins from their skeletal protein linkages leads to decreased cohesion between the bilayer and the membrane skeleton culminating in membrane loss. 2) Explore the hypothesis that lateral linkages amongst skeletal proteins in intact membranes are dynamically regulated and contribute to membrane cohesion. 3) Explore the hypothesis that direct interactions between skeletal proteins and phosphatidylserine can modulate membrane cohesion. 4) Define the genesis of red cell membrane function through characterization of the structural organization and membrane properties of reticulocyte membranes. For these studies we will make extensive use of pathologic human red cells, red cells from knockout mice and red ceils reconstituted with purified and recombinantly expressed proteins. The mechanical integrity of native and reconstituted cells will be analyzed using ektacytometric membrane fragmentation assay. The connectivity between cytoplasmic domains of various integral proteins and the membrane skeleton will be assessed using the fluorescence-imaged micropipet aspiration method and the forces involved in separation of bilayer from the membrane skeleton will be quantitated using the technique of magnetic tweezers. We anticipate that successful accomplishment of these aims, using molecular engineering and cell reconstitution methods in conjunction with novel biophysical approaches, will enable us to critically test the various hypotheses for the origins of membrane material behavior at the molecular level. The information derived should continue to provide a detailed understanding of the molecular basis for red cell membrane material properties and offer significant insights into functional consequences of membrane disorganization in pathologic red cells.